Phytoremediation is the process by which plants are used to remove pollutants like heavy metals from the environment. The roots of the plant “suck up” the pollutants from the environment and these can be stored within the plant. Plants thrive by photosynthesis, hence phytoremediation is solar-driven, environmentally-friendly, low-cost and remediation occurs in situ.
Heavy metals, the undesirable products from industries like mining and manufacturing, as well as agriculture, contaminate the environment by polluting streams, sediment, sludge, groundwater, and soil. Transgenic plants have been successfully used to detoxify heavy metals like mercury, cadmium, arsenate, and selenate from soil (Kramer, Curr. Opin. Biotech. 16: 133-141, 2005). Such heavy metals, toxic to humans and animals, adversely affect the human nervous system and induce cancers. These pollutants are also known to stress the growth and development of wild-type plants growing on contaminated soils. Transgenic plants that contain heterologous gene(s), with the ability to “detoxify” the pollutant, can tolerate the heavy metal stress and will concurrently clean-up the environment. The toxin will be absorbed and concentrated in the plant tissue such as in leaves and stems. Subsequently, these plants (if the toxin is yet not degraded) can be harvested and then incinerated safely. This is especially applicable to metal pollutants including arsenate, cadmium, and mercury which cannot be easily broken down (Powell, Nature doi: 10.1038/news021100-14, 2002).
Genetic engineering has made it possible to transfer non-plant derived genes for expression in plants. Examples in phytoremediation of genetically-transformed plants expressing non-plant genes include those that express bacterial enzymes that breakdown arsenic compounds in transgenic Arabidopsis (Dhankher et al., Nature Biotech. 20: 1140-1146, 2002) and others that detoxify mercury (Kramer, Curr. Opin. Biotech. 16: 133-141, 2005). The generation of transgenic Arabidopsis and Brassica plants that detoxify selenate has also been reported (Kramer, Curr. Opin. Biotech. 16: 133-141, 2005).
There is an apparent lack of plant genes that encode proteins capable of binding lead. Transgenic plants that can potentially phytoremediate lead have been generated by the expression of a yeast YCF1 protein (Song et al., Nature Biotech. 21: 914-919, 2003). Bacterial P-type ATPases which remove lead are deemed unsuitable for phytoremediation because their use will not culminate in the accumulation of lead in plants cells (Song et al., Nature Biotech. 21: 914-919, 2003). Since lead toxicity is of prime concern to human health, particularly that of children, phytoremediation would provide a useful strategy to eliminate lead accumulation and its concentration in food chains. Hence, such procedures in lead bioremediation are invaluable for the protection of human health and the environment worldwide.
It has been previously reported that two low molecular weight cytosolic proteins isolated from human kidney tissue have been observed to bind physiologic lead in vivo with high affinities. The two human proteins were identified as thymosin beta-4 of molecular mass 5 kDa and a 9-kDa acyl-CoA-binding protein (Smith et al., Chemico-Biological Interactions 115: 39-52, 1998). These small proteins, known to be highly-conserved in mammals, have been suggested to be the specific molecular targets for lead in environmentally-exposed humans (Smith et al., Chemico-Biological Interactions 115: 39-52, 1998).
The 9-kDa human ACBP is homologous to the bovine 10-kDa cytosolic ACBP (diazepam-binding inhibitor/enzepine), and such 10-kDa ACBPs have already been well-characterized in many organisms (reviewed in Kragelund et al., Biochim Biophys Acta 1441: 150-161, 1999) including man (Swinnen et al., DNA Cell Biol. 15: 197-208, 1996). Bovine 10-kDa ACBP and rat 10-kDa ACBP have been demonstrated to bind palmitoyl-CoA and oleoyl-CoA (Rasmussen et al., Biochem. J. 265: 849-855, 1990). The 10-kDa ACBP has been implicated to mediate intracellular acyl-CoA transport by binding long-chain acyl-CoA esters (reviewed in Kragelund et al., Biochim Biophys Acta 1441: 150-161, 1999). These long-chain acyl-CoAs esters not only function as intermediates in lipid metabolism but have been implicated in protein trafficking, vesicular trafficking, and gene regulation (reviewed in Faergman and Knudsen, Biochem. J. 323: 1-12, 1997).
In the model plant Arabidopsis, a 10-kDa ACBP (GenBank Accession No. NP—174462) that is homologous to the previously characterised human and bovine ACBPs, has been reported by Engeseth et al. (Arch. Biochem. Biophys. 331: 55-62, 1996). However, our recent work has shown that other forms of ACBPs are also known to occur in Arabidopsis (Leung et al., Plant Mol. Biol. 55: 297-309, 2004). The Arabidopsis complete ACBP gene family of six members encode proteins ranging from 92 amino acids to 668 amino acids, each containing a conserved acyl-CoA-binding domain (Leung et al., Plant Mol. Biol. 55: 297-309, 2004). Specifically, they are the Arabidopsis ACBP6 (SEQ ID NO: 26) (10-kDa ACBP, GenBank Accession No. NP—174462, Engeseth et al., Arch. Biochem. Biophys. 331: 55-62, 1996), membrane-associated ACBP1 (SEQ ID NO: 27) (GenBank Accession No. AAD03482, Chye et al., Plant J. 18: 205-214, 1999), membrane-associated ACBP2 (SEQ ID NO: 28) (GenBank Accession No. NP—194507, Chye et al., Plant Mol. Biol. 44: 711-721, 2000; Li and Chye, Plant Mol. Biol. 51: 483-492, 2003), ACBP3 (SEQ ID NO: 29) (GenBank Accession No. NP—194154, Leung et al., Planta 223: 871-881, 2006) and the two kelch-motif-containing ACBPs, ACBP4 (SEQ ID NO: 30) (GenBank Accession No. NP—187193, Leung et al., Plant Mol. Biol. 55: 297-309, 2004) and ACBP5 (SEQ ID NO: 31) (GenBank Accession No. NP—198115, Leung et al., Plant Mol. Biol. 55: 297-309, 2004).
Many ACBPs identified in other organisms are 10-kDa homologs of the 10-kDa bovine ACBP, consisting of 86-104 amino acids, or variants thereof arising from alternative first exon usage (Nitz et al., Int. J. Biochem. Cell Biol. 37: 2395-2405, 2005). The membrane-associated domains of Arabidopsis ACBP1 (consisting of 338 amino acids) and ACBP2 (consisting of 354 amino acids) are located at the N-terminus and both have C-terminal ankyrin repeats. ACBP1 and ACBP2 share 76.9% amino acid identity. Highly-conserved (81.4% identity) ACBP4 and ACBP5 both contain C-terminal kelch motifs. Ankyrin repeats and kelch motifs are domains that can potentially mediate protein-protein interactions, suggesting these Arabidopsis ACBPs can interact with protein partners (Leung et al., Plant Mol. Biol. 55: 297-309, 2004).
Thus, according to presence of structural domains, the Arabidopsis ACBP family can be divided into four classes: (1) small 10-kDa ACBP6 of 92 amino acids; (2) ACBP1 (338 amino acids) and ACBP2 (354 amino acids) with N-terminal membrane-associated domains and C-terminal ankyrin repeats; (3) ACBP3 of 362 amino acids; and (4) kelch-motif containing large ACBP4 (668 amino acids) and ACBP5 (648 amino acids). To establish the significance of each acyl-CoA-binding domain in binding acyl-CoA esters, Arabidopsis ACBPs have been expressed as recombinant (His)-tagged proteins in Escherichia coli for in vitro binding assays, and residues within the acyl-CoA-binding domain essential in binding have been identified by site-directed mutagenesis (Chye et al., Plant Mol. Biol. 44: 711-721, 2000; Leung et al., Plant Mol. Biol. 55: 297-309, 2004; Leung et al., Planta 223: 871-881, 2006). The differential binding affinities of Arabidopsis ACBPs to various acyl-CoA esters suggest they may possess different cellular functions.
Since the expression of plant-derived gene(s) in transformed plants may generally be more acceptable to the public, we provide herein below a method for the phytoremediation of lead and other metals by the expression or overexpression of plant-derived acyl-CoA-binding proteins (ACBPs) in transformed plants.